MHC Class I-restricted CD8+ T cells are an important component of the immune response to HIV as well as the simian immunodeficiency virus (SIV). It is generally accepted that a functional vaccine against HIV will need to elicit an effective CD8+ T cell response. This proposal will develop techniques to be used in the Rhesus macaque AIDS vaccine animal model to assess functional and quantitative aspects of the CD8+ T cell response to SIV. We will use established techniques including the intracellular cytokine assay along with a novel method we are developing to quantify SIV specific CD8+ T cell clonotypes in macaques. These techniques will assess the magnitude (percent of CD8+ cells), breadth (number of SIV epitopes), and the clonality (number of T cell receptors (TCRs)) of the CD8+ T-cell immune response to SIV vaccination and compare these to a natural infection. The specific aims for this proposal are as follows: 1. To identify and quantify by intracellular cytokine staining SIV specific CD8+ T cells in Rhesus macaques using peptide panels which encompass entire SIV proteins The innovative aspect of Aim 1 is that it permits an in depth analysis of the CD8+ T cell response to an entire protein but does not rely on any assumptions of immunodominance of one epitope over another and can be used in any macaque regardless of MHC haplotype. 2. To develop a TCR Molecular Clonotype Tracking Assay to identify and monitor the TCR genotypes of SIV specific CD8+ T cells in Rhesus macaques. The innovative aspect of Aim 2 is that it utilizes a combination of SIV peptide stimulation and cytokine secretion permitting an analysis of the TCR clonotypic repertoire and tracking of the responding CD8+ T cells. This will permit an in depth analysis of the clonality of SIV-specific CD8+ genotypes elicited during vaccination and a determination of whether these clonotypes expand or are eliminated after viral challenge. Utilizing these techniques requires no prior knowledge of either the epitope specificities of SIV-specific CD8+ T cells or the macaque's MHC haplotype. We can therefore proceed with these studies in macaques with the reagents we currently possess. Development of the techniques described above will provide in depth information with regard to SIV-specific CD8+ T cells elicited during vaccination or infection which cannot be obtained with current technology.